The present invention relates to novel Staphylococcus aureus genes (S. aureus) nucleic acids and polypeptides. Also provided are vectors, host cells and recombinant methods for producing the same. Further provided are diagnostic methods for detecting Staphylococcus aureus using probes, primers, and antibodies to the S. aureus nucleic acids and polypeptides of the present invention. The invention further relates to screening methods for identifying agonists and antagonists of S. aureus polypeptide activity and to vaccines using S. aureus nucleic acids and polypeptides.
The genus Staphylococcus includes at least 20 distinct species. (For a review see Novick, R. P., The Staphylococcus as a Molecular Genetic System in MOLECULAR BIOLOGY OF THE STAPHYLOCOCCI, 1-37 (R. Novick, Ed., VCH Publishers, New York (1990)). Species differ from one another by 80% or more, by hybridization kinetics, whereas strains within a species are at least 90% identical by the same measure.
The species S. aureus, a gram-positive, facultatively aerobic, clump-forming cocci, is among the most important etiological agents of bacterial infection in humans, as discussed briefly below.
Human Health and S. aureus 
Staphylococcus aureus is a ubiquitous pathogen. See, e.g., Mims et al., MEDICAL MICROBIOLOGY (Mosby-Year Book Europe Limited, London, UK 1993). It is an etiological agent of a variety of conditions, ranging in severity from mild to fatal. A few of the more common conditions caused by S. aureus infection are burns, cellulitis, eyelid infections, food poisoning, joint infections, neonatal conjunctivitis, osteomyelitis, skin infections, surgical wound infection, scalded skin syndrome and toxic shock syndrome, some of which are described further below.
Burns: Burn wounds generally are sterile initially. However, they generally compromise physical and immune barriers to infection, cause loss of fluid and electrolytes and result in local or general physiological dysfunction. After cooling, contact with viable bacteria results in mixed colonization at the injury site. Infection may be restricted to the non-viable debris on the burn surface (xe2x80x9cescharxe2x80x9d), it may progress into full skin infection and invade viable tissue below the eschar and it may reach below the skin, enter the lymphatic and blood circulation and develop into septicemia. S. aureus is among the most important pathogens typically found in burn wound infections. It can destroy granulation tissue and produce severe septicemia.
Cellulitis: Cellulitis, an acute infection of the skin that expands from a typically superficial origin to spread below the cutaneous layer, most commonly is caused by S. aureus in conjunction with S. pyrogenes. Cellulitis can lead to systemic infection. In fact, cellulitis can be one aspect of synergistic bacterial gangrene. This condition typically is caused by a mixture of S. aureus and microaerophilic streptococci. It causes necrosis and treatment is limited to excision of the necrotic tissue. The condition often is fatal.
Eyelid infections: S. aureus is the cause of styes and of sticky eyexe2x80x9d in neonates, among other eye infections. Typically such infections are limited to the surface of the eye, and may occasionally penetrate the surface with more severe consequences.
Food poisoning: Some strains of S. aureus produce one or more of five serologically distinct, heat and acid stable enterotoxins that are not destroyed by digestive process of the stomach and small intestine (enterotoxins A-E). Ingestion of the toxin, in sufficient quantities, typically results in severe vomiting, but not diarrhea. The effect does not require viable bacteria. Although the toxins are known, their mechanism of action is not understood.
Joint infections: S. aureus infects bone joints causing diseases such osteomyelitis. See, e.g., R. Cunningham et al., (1996) J. Med. Microbiol. 44:157-164.
Osteomyelitis: S. aureus is the most common causative agent of haematogenous osteomyelitis. The disease tends to occur in children and adolescents more than adults and it is associated with non-penetrating injuries to bones. Infection typically occurs in the long end of growing bone, hence its occurrence in physically immature populations. Most often, infection is localized in the vicinity of sprouting capillary loops adjacent to epiphysis growth plates in the end of long, growing bones.
Skin infections: S. aureus is the most common pathogen of such minor skin infections as abscesses and boils. Such infections often are resolved by normal host response mechanisms, but they also can develop into severe internal infections. Recurrent infections of the nasal passages plague nasal carriers of S. aureus. 
Surgical Wound Infections: Surgical wounds often penetrate far into the body. Infection of such wound thus poses a grave risk to the patient. S. aureus is the most important causative agent of infections in surgical wounds. S. aureus is unusually adept at invading surgical wounds; sutured wounds can be infected by far fewer S. aureus cells then are necessary to cause infection in normal skin. Invasion of surgical wound can lead to severe S. aureus septicemia. Invasion of the blood stream by S. aureus can lead to seeding and infection of internal organs, particularly heart valves and bone, causing systemic diseases, such as endocarditis and osteomyelitis.
Scalded Skin Syndrome: S. aureus is responsible for xe2x80x9cscalded skin syndromexe2x80x9d (also called toxic epidermal necrosis, Ritter""s disease and Lyell""s disease). This diseases occurs in older children, typically in outbreaks caused by flowering of S. aureus strains produce exfoliation (also called scalded skin syndrome toxin). Although the bacteria initially may infect only a minor lesion, the toxin destroys intercellular connections, spreads epidermal layers and allows the infection to penetrate the outer layer of the skin, producing the desquamation that typifies the diseases. Shedding of the outer layer of skin generally reveals normal skin below, but fluid lost in the process can produce severe injury in young children if it is not treated properly.
Toxic Shock Syndrome: Toxic shock syndrome is caused by strains of S. aureus that produce the so-called toxic shock syndrome toxin. The disease can be caused by S. aureus infection at any site, but it is too often erroneously viewed exclusively as a disease solely of women who use tampons. The disease involves toxemia and septicemia, and can be fatal.
Nocosomial Infections: In the 1984 National Nocosomial Infection Surveillance Study (xe2x80x9cNNISxe2x80x9d) S. aureus was the most prevalent agent of surgical wound infections in many hospital services, including medicine, surgery, obstetrics, pediatrics and newborns.
Other Infections: Other types of infections, risk factors, etc. involving S. aureus are discussed in: A. Trilla (1995) J. Chemotherapy 3:37-43; F. Espersen (1995) J. Chemotherapy 3:11-17; D. E. Craven (1995) J. Chemotherapy 3:19-28; J. D. Breen et al. (1995) Infect. Dis. Clin. North Am. 9(1):11-24 (each incorporated herein in their entireties).
Resistance to Drugs of S. aureus Strains
Prior to the introduction of penicillin the prognosis for patients seriously infected with S. aureus was unfavorable. Following the introduction of penicillin in the early 1940s even the worst S. aureus infections generally could be treated successfully. The emergence of penicillin-resistant strains of S. aureus did not take long, however. Most strains of S. aureus encountered in hospital infections today do not respond to penicillin; although, fortunately, this is not the case for S. aureus encountered in community infections.
It is well known now that penicillin-resistant strains of S. aureus produce a lactamase which converts penicillin to pencillinoic acid, and thereby destroys antibiotic activity. Furthermore, the lactamase gene often is propagated episomally, typically on a plasmid, and often is only one of several genes on an episomal element that, together, confer multidrug resistance.
Methicillins, introduced in the 1960s, largely overcame the problem of penicillin resistance in S. aureus. These compounds conserve the portions of penicillin responsible for antibiotic activity and modify or alter other portions that make penicillin a good substrate for inactivating lactamases. However, methicillin resistance has emerged in S. aureus, along with resistance to many other antibiotics effective against this organism, including aminoglycosides, tetracycline, chloramphenicol, macrolides and lincosamides. In fact, methicillin-resistant strains of S. aureus generally are multiply drug resistant.
Methicillian-resistant S. aureus (MRSA) has become one of the most important nosocomial pathogens worldwide and poses serious infection control problems. Today, many strains are multiresistant against virtually all antibiotics with the exception of vancomycin-type glycopeptide antibiotics.
Recent reports that transfer of vancomycin resistance genes from enterococci to S. aureus has been observed in the laboratory sustain that fear that MRSA might become resistant against vancomycin, too, a situation generally considered to result in a public health disaster. MRSA owe their resistance against virtually all xcex2-lactam antibiotics to the expression of an extra penicillin binding protein (PBP) 2a, encoded by the mecA gene. This additional very low affinity pbp, which is found exclusively in resistant strains, appears to be the only pbp still functioning in cell wall peptidoglycan synthesis at xcex2-lactam concentrations high enough to saturate the normal set of S. aureus pbp 1-4. In 1983 it was shown by insertion mutagenesis using transposon Tn551 that several additional genes independent of mecA are needed to sustain the high level of methicillin resistance of MRSA. Interruption of these genes did not influence the resistance level by interfering with PBP2a expression, and were therefore called fem (factor essential for expression of methicillin resistance) or aux (auxiliary genes).
In the meantime six fem genes (femA- through F) have been described and the minimal number of additional aux genes has been estimated to be more than 10. Interference with fema and femB results in a strong reduction of methicillin resistance, back to sensitivity of strains without PBP2a. The fem genes are involved in specific steps of cell wall synthesis. Consequently, inactivation of fem factors induce xcex2-lactam hypersensitivity in already sensitive strains. Both femA and femB have been shown to be involved in peptidoglycan pentaglycine interpeptide bridge formation. FemA is responsible for the formation of glycines 2 and 3, and femB is responsible for formation of glycines 4 and 5. S. aureus may be involved in the formation of a monoglycine muropeptide precursors. FemC-F influence amidation of the iso-D-glutamic acid residue of the peptidoglycan stem peptide, formation of a minor muropeptide with L-alanine instead of glycine at position 1 of the interpeptide bridge, perform a yet unknown function, or are involved in an early step of peptidoglycan precursors biosynthesis (addition of L-lysine), respectively.
Thus far each new antibiotic gives rise to resistance strains, emerge that are resistance to multiple drugs and increasingly persistent forms of resistance begin to emerge. Drug resistance of S. aureus infections already poses significant treatment difficulties, which are likely to get much worse unless new therapeutic agents are developed. Since S. aureus is likely involved in the synthesis of the peptidoglycan cross bridges in S. aureus, the gene provides an important tool in studying the mechanisms of antibiotic resistance. The S. aureus gene and its polypeptides are also potential target for antagonists or agonists, which may be useful as antibiotics, or useful to block resistance to other antibiotics. That is, antagonists or agonists, such as small molecules, may be useful as antibiotics themselves, act additively with other antibiotics, or act synergistically with other antibiotics.
The present invention provides isolated S. aureus polynucleotides and polypeptides shown in Table 1 and SEQ ID NO:1 through SEQ ID NO:22 (polynucleotide sequences having odd SEQ ID NOs and polypeptide sequences having even SEQ ID NOs). One aspect of the invention provides isolated nucleic acid molecules comprising polynucleotides having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence shown in Table 1; (b) a nucleotide sequence encoding any of the amino acid sequences of the polypeptides shown in Table 1; and (c) a nucleotide sequence complementary to any of the nucleotide sequences in (a) or (b). The invention further provides for fragments of the nucleic acid molecules of (a), (b) and (c) above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b) or (c) above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b) or (c) above. Additional nucleic acid embodiments of the invention relate to isolated nucleic acid molecules comprising polynucleotides which encode the amino acid sequences of epitope-bearing portions of a S. aureus polypeptide having an amino acid sequence in (a) above.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells. The present invention further relates to the use of these vectors in the production of S. aureus polypeptides or peptides by recombinant techniques.
The invention further provides isolated S. aureus polypeptides having an amino acid sequence selected from the group consisting of an amino acid sequence of any of the polypeptides described in Table 1 or fragments thereof.
The polypeptides of the present invention also include polypeptides having an amino acid sequence with at least 70% similarity, and more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similarity to those described in Table 1, as well as polypeptides having an amino acid sequence at least 70% identical, more preferably at least 75% identical, and still more preferably 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to those above; as well as isolated nucleic acid molecules encoding such polypeptides.
The present invention further provides a vaccine, preferably a multi-component vaccine comprising one or more of the S. aureus polynucleotides or polypeptides described in Table 1, or fragments thereof, together with a pharmaceutically acceptable diluent, carrier, or excipient, wherein the S. aureus polypeptide(s) are present in an amount effective to elicit an immune response to members of the Staphylococcus genus, or at least S. aureus, in an animal. The S. aureus polypeptides of the present invention may further be combined with one or more immunogens of one or more other staphylococcal or non-staphylococcal organisms to produce a multi-component vaccine intended to elicit an immunological response against members of the Staphylococcus genus and, optionally, one or more non-staphylococcal organisms.
The vaccines of the present invention can be administered in a DNA form, e.g., xe2x80x9cnakedxe2x80x9d DNA, wherein the DNA encodes one or more staphylococcal polypeptides and, optionally, one or more polypeptides of a non-staphylococcal organism. The DNA encoding one or more polypeptides may be constructed such that these polypeptides are expressed as fusion proteins.
The vaccines of the present invention may also be administered as a component of a genetically engineered organism or host cell. Thus, a genetically engineered organism or host cell which expresses one or more S. aureus polypeptides may be administered to an animal. For example, such a genetically engineered organism or host cell may contain one or more S. aureus polypeptides of the present invention intracellularly, on its cell surface, or in its periplasmic space. Further, such a genetically engineered organism or host cell may secrete one or more S. aureus polypeptides. The vaccines of the present invention may also be co-administered to an animal with an immune system modulator (e.g., CD86 and GM-CSF).
The invention also provides a method of inducing an immunological response in an animal to one or more members of the Staphylococcus genus, preferably one or more isolates of the S. aureus species, comprising administering to the animal a vaccine as described above.
The invention further provides a method of inducing a protective immune response in an animal, sufficient to prevent, attenuate, or control an infection by members of the Staphylococcus genus, preferably at least S. aureus species, comprising administering to the animal a composition comprising one or more of the polynucleotides or polypeptides described in Table 1, or fragments thereof. Further, these polypeptides, or fragments thereof, may be conjugated to another immunogen and/or administered in admixture with an adjuvant.
The invention further relates to antibodies elicited in an animal by the administration of one or more S. aureus polypeptides of the present invention and to methods for producing such antibodies and fragments thereof. The invention further relates to recombinant antibodies and fragments thereof and to methods for producing such antibodies and fragments thereof.
The invention also provides diagnostic methods for detecting the expression of the polynucleotides of Table 1 by members of the Staphylococcus genus in an animal. One such method involves assaying for the expression of a polynucleotide encoding S. aureus polypeptides in a sample from an animal. This expression may be assayed either directly (e.g., by assaying polypeptide levels using antibodies elicited in response to amino acid sequences described in Table 1) or indirectly (e.g., by assaying for antibodies having specificity for amino acid sequences described in Table 1). The expression of polynucleotides can also be assayed by detecting the nucleic acids of Table 1. An example of such a method involves the use of the polymerase chain reaction (PCR) to amplify and detect Staphylococcus nucleic acid sequences.
The present invention also relates to nucleic acid probes having all or part of a nucleotide sequence described in Table 1 (odd SEQ ID NOs) which are capable of hybridizing under stringent conditions to Staphylococcus nucleic acids. The invention further relates to a method of detecting one or more Staphylococcus nucleic acids in a biological sample obtained from an animal, said one or more nucleic acids encoding Staphylococcus polypeptides, comprising: (a) contacting the sample with one or more of the above-described nucleic acid probes, under conditions such that hybridization occurs, and (b) detecting hybridization of said one or more probes to the Staphylococcus nucleic acid present in the biological sample.
Polynucleotides and Polypeptides of the Invention
Features of femX Polynucleotides and Polypeptides.
The nucleotide sequence shown in SEQ ID NO:1 was determined by sequencing the S. aureus overlapping clones BTEFS71 and BTEJE39. The nucleotide sequence contains an open reading frame encoding the femX polypeptide comprising 414 amino acid residues (SEQ ID NO:2), including an initiation codon encoding an N-terminal methionine at nucleotide positions 164-166, and a predicted molecular weight of about 49.1 kDa.
The femX polypeptides of the present invention have amino acid sequence homology to known genes involved in formation of peptidoglycan cross bridges, including the conserved cysteine pattern characteristic of the epr and fem family of genes. The S. aureus femX polypeptide of SEQ ID NO:2 was found to share a high degree of local sequence identity with amino acid sequences of the epr (M. Sugai, et al. (1997) J. Bacteriol. 179(13):4311-4318) and fem A and fem B proteins from Staphlococcus species (A. M. Stranden et al., (1997) J. Bacteriol. 179(1):9-16; G. Thumm et al. (1997) Mol. Microbiol. 23(6):1251-1265; W. E. Alborn et al., (1996) Gene 180(1-2):177-181) using the computer program BLAST (Altschul et al., (1990) J. Mol. Biol. 215:403-410).
The strong homology between species and identity among fem proteins of S. aureus indicates that femX is involved in peptidoglycan interpeptide bridge biosynthesis. Thus, the polypeptides of the present invention are useful in screening methods to make antagonists which block their function. Antagonists can be used, for instance, as antibiotics to treat antibiotic resistant S. aureus or other Staphylococcus species. Antagonists of the polypeptides of the present invention can be identified by measuring the formation of peptidoglycan cross bridges. More specifically, the synthesis of glycines 1-5 of peptidoglycan cross bridges can be measured as exemplified by A. M. Stranden et al. (1997) J. Bacteriol 179(1):9-16 (incorporated herein in its entirety). Antagonists of femX would act to inhibit peptidoglycan cross bridge formation.
Other uses of the femX polypeptides of the present invention include: inter alia, to detect S. aureus in immunoassays, as epitope tags, as molecular weight markers on SDS-PAGE gels, as molecular weight markers for molecular sieve gel filtration columns, to generate antibodies that specifically bind S. aureus femX for the detection S. aureus in immunoassays, to generate an immune response against S. aureus and other Staphylococcus species, and as vaccines against S. aureus and other Staphylococcus species.
Isolated nucleic acid molecules of the present invention, particularly DNA molecules, are useful as probes for gene mapping and for identifying S. aureus in a biological samples, for instance, by Southern and Northern blot analysis. femX polynucleotides of the present invention are also useful in detecting S. aureus by PCR using primers for femX polynucleotides. Isolated polynucleotides of the present invention are also useful in making the polypeptides of the present invention.
Features of furA, furB, and furC Polynucleotides and Polypeptides.
The nucleotide sequences for furA, furB, and furC were determined by sequencing the S. aureus clones BTEJQ50 (SEQ ID NO:3), BTALE70 (SEQ ID NO:5), and BTEBP80 and BTEFD68 (collectively SEQ ID NO:7). The nucleotide sequence of SEQ ID NO:3 contains an open reading frame encoding the furA polypeptide comprising 136 amino acid residues (SEQ ID NO:4), including an initiation codon encoding an N-terminal methionine at nucleotide positions 101-103, and a predicted molecular weight of about 15.9 kDa.
The nucleotide sequence of SEQ ID NO:5 contains an open reading frame encoding the furB polypeptide comprising 148 amino acid residues (SEQ ID NO:6), including an initiation codon encoding an N-terminal methionine at nucleotide positions 101-103, and a predicted molecular weight of about 17.2 kDa.
The nucleotide sequence of SEQ ID NO:7 contains an open reading frame encoding the furC polypeptide comprising 149 amino acid residues (SEQ ID NO:8), including an initiation codon encoding an N-terminal leucine at nucleotide positions 101-103, and a predicted molecular weight of about 17.2 kDa.
The fur (ferric uptake regulator) polypeptides (furA, furB, and furC) of the present invention have amino acid sequence homology to known genes involved in iron regulation. The S. aureus furA polypeptide of SEQ ID NO:2 was found to share a high degree of local sequence identity with the amino acid sequence of a fur gene from Staphylococcus epidermidis (GenBank accession number gnl|PID|e236389). See C. Heidrich et al. (1996) FEMS Micro. Letts. 140:253-259. The S. aureus furB polypeptide of SEQ ID NO:2 was found to share a high degree of local sequence identity with the amino acid sequence of a fur family gene from Bacillus subtilis (GenBank accession number gnl|PID|e281583). See N. J Cummings et al. (1997) Microbiology 143:1855-1859. The S. aureus furC polypeptide of SEQ ID NO:2 was found to share a high degree of local sequence identity with the amino acid sequence of another fur family gene from Bacillus subtilis (GenBank accession number gnl|PID|e1185621). See F. Kunst et al. (1997) Nature 390:249-256.
The fur polypeptides of the present invention also share identity among themselves as well as other fur and fur-like genes from Bacillus subtilis (GenBank accession numbers gnl|PID|e1185777), Streptococcus pyogenes (GenBank accession number gi|1667516), Neisseria meningitidis (GenBank accession number gi|433299), Neisseria gonorrheae (GenBank accession number gi|349012), Camplyobacter upsaliensis (GenBank accession number gi|1228779), Camplyobacter jejuni (GenBank accession number gi|511113) Mycobacterium tuberculosis (GenBank accession numbergnl|PID|e315163), and other bacteria species. Identities were compared using the computer program BLAST (Altschul et al., (1990) J. Mol. Biol. 215:403-410).
The strong homology among the fur proteins of S. aureus and other bacteria species indicates that furA, furB, and furC are involved in iron regulation in S. aureus. Since iron is essential for the growth and multiplication of nearly microorganisms, the polypeptides of the present invention are useful in screening methods to make antagonists which block their function. Antagonists can be used, for instance, as antibiotics to treat infections of S. aureus or other Staphylococcus species. Antagonists of the polypeptides of the present invention can be identified by measuring the ability of bacteria to grow in the presence of varying concentrations of iron.
Other uses of the fur polypeptides of the present invention include: inter alia, to detect S. aureus in immunoassays, as epitope tags, as molecular weight markers on SDS-PAGE gels, as molecular weight markers for molecular sieve gel filtration columns, to generate antibodies that specifically bind S. aureus furA, furB, and furC for the detection S. aureus in immunoassays, to generate an immune response against S. aureus and other Staphylococcus species, and as vaccines against S. aureus, other Staphylococcus species and other bacteria genuses.
Isolated nucleic acid molecules of the present invention, particularly DNA molecules, are useful as probes for gene mapping and for identifying S. aureus in a biological samples, for instance, by Southern and Northern blot analysis. fur polynucleotides of the present invention are also useful in detecting S. aureus by PCR using primers for a particular fur polynucleotide. Isolated polynucleotides of the present invention are also useful in making the polypeptides of the present invention.
Features of fmtB, pbpF, and pbpG Polynucleotides and Polypeptides.
The nucleotide sequences for fmtB, pbpF, and pbpG were determined by sequencing the S. aureus clones BTEDA22 and BTEDV18 (SEQ ID NO:9), BTEBG73 and BTAJO70 (SEQ ID NO:11), and BTEBU53 and BTEFB55 (SEQ ID NO:13), respectively. The nucleotide sequence of SEQ ID NO:9 contains an open reading frame encoding the fmtB polypeptide comprising 498 amino acid residues (SEQ ID NO:10), including an initiation codon encoding an N-terminal methionine at nucleotide positions 101-103, and a predicted molecular weight of about 56.4 kDa.
The nucleotide sequence of SEQ ID NO:11 contains an open reading frame encoding the pbpF polypeptide comprising 691 amino acid residues (SEQ ID NO:12), including an initiation codon encoding an N-terminal leucine at nucleotide positions 101-103, and a predicted molecular weight of about 77.2 kDa.
The nucleotide sequence of SEQ ID NO:13 contains an open reading frame encoding the pbpG polypeptide comprising 301 amino acid residues (SEQ ID NO:14), including an initiation codon encoding an N-terminal methionine at nucleotide positions 101-103, and a predicted molecular weight of about 34.5 kDa.
The fmtB, pbpF, and pbpG polypeptides of the present invention have amino acid sequence homology to known penicillin-biding proteins among several species. The S. aureus fmtB polypeptide of SEQ ID NO:10 was found to share local sequence identity, inter alia, with the amino acid sequence of a penicillin-binding protein gene from Bacillus subtilis (GenBank accession number gnl|PID|e1185286). See F. Kunst et al. (1997) Nature 390:249-256. fmtB also shares sequence identity with another Staphylococcus aureus polypeptide associated with antibiotic resistance (GenBank accession number gnl|PID|d1024918). See H. Komatsuzawa et al. (1997) Antimicrob. Agents Chemother. 41:2355-2361.
The S. aureus pbpF polypeptide of SEQ ID NO:12 was found to share local sequence identity, inter alia, with the amino acid sequence of penicillin-binding genes from Bacillus subtilis (GenBank accession number gnl|PID|e1181903 and gnl|PID|e185767) and Streptococcus thermophilus (GenBank accession number gi|643510). The S. aureus pbpG polypeptide of SEQ ID NO:14 was found to share local sequence identity, inter alia, with the amino acid sequence of penicillin-binding genes from Pseudomonas syringae (GenBank accession number gi|551940). See E. Roine et al. (1996) J. Bacteriol. 178:410-417, and Bacillus subtilis (GenBank accession number gnl|PID|e267588). Identities were compared using the computer program BLAST (Altschul et al, (1990) J. Mol. Biol. 215:403-410).
The strong homology among the S. aureus fmtB, pbpF, and pbpG polypeptides of the present invention and penicillin-binding proteins from other bacteria species indicates that fmtB, pbpF, and pbpG are involved cell wall synthesis and in xcex2-lactam resistance in S. aureus. The polypeptides of the present invention are therefore useful for making compounds that inhibit their function for use as antibiotics. Inhibitors of the polypeptides of the present invention can be identified by measuring the ability of bacteria to grow in the presence of varying concentrations of antibiotics or by cell wall synthesis assays using methods known in the art.
Other uses of the fmtB, pbpF, and pbpG polypeptides of the present invention include: inter alia, to detect S. aureus in immunoassays, as epitope tags, as molecular weight markers on SDS-PAGE gels, as molecular weight markers for molecular sieve gel filtration columns, to generate antibodies that specifically bind S. aureus fmtB, pbpF, or pbpG for the detection S. aureus in immunoassays, to generate an immune response against S. aureus and other Staphylococcus species, and as vaccines against S. aureus, other Staphylococcus species and other bacteria genuses.
Isolated nucleic acid molecules of the present invention, particularly DNA molecules, are useful as probes for gene mapping and for identifying S. aureus in a biological samples, for instance, by Southern and Northern blot analysis. fmtB, pbpF, and pbpG polynucleotides of the present invention are also useful in detecting S. aureus by PCR using primers for a particular fmtB, pbpF, or pbpG polynucleotide. Isolated polynucleotides of the present invention are also useful in making the polypeptides of the present invention.
Features of cbrA, cbrB, and cbrC Polynucleotides and Polypeptides.
The nucleotide sequences for cbrA (SEQ ID NO:15), cbrB (SEQ ID NO:17), and cbrC (SEQ ID NO:19) comprise a single operon and were determined by sequencing the S. aureus overlapping clones BTACA44 and BTAGJ54 which span the operon. The nucleotide sequence of SEQ ID NO:15 contains an open reading frame encoding the cbrA polypeptide comprising 330 amino acid residues (SEQ ID NO:16), including an initiation codon encoding an N-terminal methionine at nucleotide positions 7-9, and a predicted molecular weight of about 36.8 kDa.
The nucleotide sequence of SEQ ID NO:17 contains an open reading frame encoding the cbrB polypeptide comprising 331 amino acid residues (SEQ ID NO:18), including an initiation codon encoding an N-terminal leucine at nucleotide positions 19-21, and a predicted molecular weight of about 35.5 kDa.
The nucleotide sequence of SEQ ID NO:19 contains an open reading frame encoding the cbrC polypeptide comprising 332 amino acid residues (SEQ ID NO:20), including an initiation codon encoding an N-terminal methionine at nucleotide positions 91-93, and a predicted molecular weight of about 35.7 kDa.
The cbr polypeptides (cbrA, cbrB, and cbrC) of the present invention have amino acid sequence homology to known genes involved in iron regulation. The S. aureus cbrA (SEQ ID NO:16), cbrB (SEQ ID NO:18), and cbrC (SEQ ID NO:20) polypeptides were found to share local sequence identity among themselves and with the amino acid sequence of a cbrA, cbrB, and cbrC genes from Erwinia chrysanthemi (GenBank accession numbers gi|809541, gi|809542, and gi|809541 respectively). See B. Mahe et al. (1995) Mol. Microbiol. 18:33-43. The cbrA, cbrB, and cbrC polypeptides of the present invention also share sequence identity and with iron regulatory genes of other bacterial species including Bacillus subtilis (GenBank accession number gnl|PID|e1182834, gnl|PID|e1182835, and gnl|PID|e1182836). See F. Kunst et al. (1997) Nature 390:249-256 and Bacillus intermedius (GenBank accession number gnl|PID|e245932). Identities were compared using the computer program BLAST (Altschul et al., (1990) J. Mol. Biol. 215:403-410).
The strong homology among the cbr proteins of S. aureus and other bacteria species indicates that cbrA, cbrB, and cbrC are involved in iron regulation in S. aureus. Since iron is essential for the growth and multiplication of nearly microorganisms, the polypeptides of the present invention are useful in screening methods to make antagonists which block their function. Antagonists can be used, for instance, as antibiotics to treat infections of S. aureus or other Staphylococcus species. Antagonists of the polypeptides of the present invention can be identified by measuring the ability of bacteria to grow in the presence of varying concentrations of iron.
Other uses of the polypeptides of the present invention include: inter alia, to detect S. aureus in immunoassays, as epitope tags, as molecular weight markers on SDS-PAGE gels, as molecular weight markers for molecular sieve gel filtration columns, to generate antibodies that specifically bind S. aureus polypeptides of the present invention for the detection S. aureus in immunoassays, to generate an immune response against S. aureus and other Staphylococcus species, and as vaccines against S. aureus, other Staphylococcus species and other bacteria genuses.
Isolated nucleic acid molecules of the present invention, particularly DNA molecules, are useful as probes for gene mapping and for identifying S. aureus in a biological samples, for instance, by Southern and Northern blot analysis. S. aureus polynucleotides of the present invention are also useful in detecting S. aureus by PCR using primers for a particular S. aureus polynucleotide. Isolated polynucleotides of the present invention are also useful in making the polypeptides of the present invention.
Features of Enolase Polynucleotides and Polypeptides.
The nucleotide sequence shown in SEQ ID NO:21 was determined by sequencing the S. aureus overlapping clones BTAAI44 and BTAGE12. The nucleotide sequence contains an open reading frame encoding the enolase polypeptide comprising 434 amino acid residues (SEQ ID NO:22), including an initiation codon encoding an N-terminal methionine at nucleotide positions 103-105, and a predicted molecular weight of about 47.1 kDa.
The enolase polypeptides of the present invention have amino acid sequence identity homology to known enolase genes from other bacterial species. The S. aureus enolase polypeptide of SEQ ID NO:22 shares local sequence identity with amino acid sequences of the enolase genes from other bacterial species including Bacillus subtilis (GenBank accession numbers gi|460259 and gnl|PID|e1186078), Spongilla sp. (GenBank accession number gi|1839206), Mycobacterium tuberculosis (GenBank accession number gnl|PID|e304557) Methanococcus jannaschii (GenBank accession number gi|1590967) and Campylobacter jejuni (GenBank accession number gi|437277).
The S. aureus enolase protein of the present invention was identified as a molecule involved in laminin (LN)/laminin receptor (LNRec) interactions. The S. aureus enolase protein of the present invention was shown to be responsible for LNRec activity in bridging experiments between the S. aureus and MDCK cell in culture. The S. aureus enolase gene of the present invention was cloned by first generating monoclonal antibodies against the LNRec molecule and using the antibodies to subsequently isolated the LNRec molecule. The LNRec molecule was purified and partially sequenced. Partial amino acid sequence analysis was used to clone and isolate the S. aureus enolase gene of the present invention. A characteristic feature of infection by S. aureus is bloodstream invasion and widespread metastatic abscess formation. The S. aureus enolase polypeptides of the present invention therefore represent a target for both vaccines and antibiotics. Antibiotics of the present invention include peptides, polypeptides, antibodies (and fragments thereof), small molecules, and other drugs that bind the S. aureus enolase polypeptides of the present invention, or enolase associated molecules, and prevent binding of S. aureus to laminin. The blocking molecules of the present invention may act by directly blocking the binding of enolase polypeptides to laminin or enolase associated molecules to laminin. Assays for measuring the binding of molecules to enolase polypeptides or enolase associated molecules; assays for measuring the binding of S. aureus to laminin; and assays for measuring the metastatic activity of S. aureus include those described and referenced in Lopes et al. (1985) Science 229:275-277, described and referenced in Brentani (1989) Oncogenesis 1:247-260, known in the art, and disclosed herein.
The structural homology and identity between the enolase polypeptides of S. aureus and those of other bacterial species indicates that the enolase polypeptides of S. aureus share the same function as the enolase polypeptides from other bacterial enolase polypeptides, including those described by Babbitt et al. (1996) Biochemistry 35:16489-16501. The enolase polynucleotides and polypeptides of the present invention are useful to produce mutant S. aureus enolase genes, polypeptides, and mutant S. aureus strains for use as vaccines and to induce an immune response in humans and other animals by using the methods described in U.S. Pat. Nos. 5,703,219 and 5,703,219. In the methods of U.S. Pat. Nos. 5,703,219 and 5,703,219 the S. aureus enolase polypeptides and polypeptides are substituted for the Helicobacter pyiori enolase polypeptides and polypeptides. Modifications to accommodate the S. aureus enolase polypeptides and polypeptides of the present invention are made using information disclosed herein and known in the art.
Other uses of the enolase polypeptides of the present invention include: inter alia, to detect S. aureus in immunoassays, as epitope tags, as molecular weight markers on SDS-PAGE gels, as molecular weight markers for molecular sieve gel filtration columns, to generate antibodies that specifically bind S. aureus enolase for the detection S. aureus in immunoassays, to generate an immune response against S. aureus and other Staphylococcus species, and as vaccines against S. aureus and other Staphylococcus species as discussed above.
Isolated nucleic acid molecules of the present invention, particularly DNA molecules, are useful as probes for gene mapping and for identifying S. aureus in a biological samples, for instance, by Southern and Northern blot analysis. enolase polynucleotides of the present invention are also useful in detecting S. aureus by PCR using primers for enolase polynucleotides. Isolated polynucleotides of the present invention are also useful in making the polypeptides of the present invention.